Genome-Wide Investigation and Expression Analysis of the Catalase Gene Family in Oat Plants (Avena sativa L.)

Through the degradation of reactive oxygen species (ROS), different antioxidant enzymes, such as catalase (CAT), defend organisms against oxidative stress. These enzymes are crucial to numerous biological functions, like plant development and defense against several biotic and abiotic stresses. However, despite the major economic importance of Avena sativa around the globe, little is known about the CAT gene’s structure and organization in this crop. Thus, a genome-wide investigation of the CAT gene family in oat plants has been carried out to characterize the potential roles of those genes under different stressors. Bioinformatic approaches were used in this study to predict the AvCAT gene’s structure, secondary and tertiary protein structures, physicochemical properties, phylogenetic tree, and expression profiling under diverse developmental and biological conditions. A local Saudi oat variety (AlShinen) was used in this work. Here, ten AvCAT genes that belong to three groups (Groups I–III) were identified. All identified CATs harbor the two conserved domains (pfam00199 and pfam06628), a heme-binding domain, and a catalase activity motif. Moreover, identified AvCAT proteins were located in different compartments in the cell, such as the peroxisome, mitochondrion, and cytoplasm. By analyzing their promoters, different cis-elements were identified as being related to plant development, maturation, and response to different environmental stresses. Gene expression analysis revealed that three different AvCAT genes belonging to three different subgroups showed noticeable modifications in response to various stresses, such as mannitol, salt, and ABA. As far as we know, this is the first report describing the genome-wide analysis of the oat catalase gene family, and these data will help further study the roles of catalase genes during stress responses, leading to crop improvement.


Introduction
Environmental stresses can considerably affect crops by more than 50% and this by affecting morphological and physiological changes in plants, which can have an impact on yield in addition to genetic factors [1].In fact, biotic and abiotic stressors have a negative impact on crop yields, biomass output, and survival [2].In general, oxidative stress is a secondary stress caused by the generation of reactive oxygen species (ROS) that occurs during aerobic cellular processes and causes an imbalance between ROS production and removal, which increases free radicals in cells [3].ROS are generated by different compartments in Plants 2023, 12, 3694 2 of 26 cells, specifically the peroxisomes, mitochondria, the endoplasmic reticulum, and chloroplasts.According to several studies [4,5], 1-2% of the oxygen a plant consumes creates ROS.At low concentrations, ROS fundamentally act as second messengers in intracellular signaling cascades of stress responses, whereas at high concentrations, ROS accumulation damages cells [6].Plant antioxidant systems can regulate ROS accumulation by controlling it synthesis under normal aerobic metabolism.This redox balance is disrupted under various stress situations by high ROS levels, depletion of antioxidant defense systems, or both, causing plant cell collapse and cell death (necrosis) [7].Biomolecules, including DNA, proteins, and lipids, can become damaged when the level of ROS surpasses the body's ability to defend itself [4].
A significant plant adaptation method to combat oxidative stress is activating several enzymatic and non-enzymatic antioxidants [8].In fact, a variety of enzymes are stimulated to lessen the effects of oxidative stress.These include ascorbate (APX), glutathione (GH) cycle enzymes, catalase (CAT), and superoxide dismutase (SOD).Almost all aerobic organisms have the antioxidant enzyme CAT, which has a high affinity for H 2 O 2 .In peroxisomes, hydrogen peroxide (H 2 O 2 ) is converted into water and oxygen [9].
A vital and well-established cereal crop, oats (Avena sativa L.) are grown essentially throughout North America and Europe [30].Actually, oats are grown worldwide as feed because of their high protein and vital mineral content [31].Additionally, it is a good source of dietary fiber, particularly glucan, which may benefit human health [32].This crop is frequently grown in regions with many drawbacks, like drought or severe salt, because it is less profitable than maize, soybean, or wheat crops [33].
This species is also sensitive to light and temperature variations [34].Because the genomes of the most significant crops, like rice [35], maize [36], and wheat [37], were sequenced, it was possible to further understand these species, but the current understanding of the stress-adaptive mechanisms in oats is still limited on a molecular level.Moreover, oats can be grown as a hay crop, cover crop, pasture plant, or green manure.It acts as a cover crop, enhancing soil health, weed control, erosion reduction, and soil organic content [38].All of this has raised the demand for high-quality oats.Due to the high capacity of oat farming to retain salt ions in its straw biomass, it is an efficient biological method to improve salty lands [39].
Cultivated oat is an allohexaploid species (AACCDD, 2n = 6x = 42) [40] thought to have been domesticated more than 3000 years ago while growing as a weed in wheat, emmer, and barley fields in Anatolia [41].This hexaploid species (6x = 42) adapts to several soil types and prospers best in cool, humid climates [42].In nature, Avena species are found as diploids, tetraploids, and hexaploids and represent the most important genetic diversity around the Canary Islands, the Mediterranean, the Middle East, and the Himalayas [41].Oat plants are more pH adaptable than wheat or barley, ranging from 5.5 to 7.0 and even as low as 4.5 for some ecotypes.Oats, however, need enough water for growth and grain production.Oats have long been superior to animal feed due to their high protein and important mineral content [38].Due to disease resistance and low nutrient needs, oat plantations also have a relatively low input need for pesticides, fungicides, and fertilizers.Although oats are primarily used as animal feed now, they are one of the cereals with the greatest potential for usage in functional foods in the future.
According to Willenborg et al. [43], salinity affects seed germination, growth, water/nutrient intake, and oat plant physiological, morphological, and biochemical processes.Firstly, we collected the gene structure information of AvCATs in the gene annotation file and visualized it using the web tool (Figure 1A).Ten CAT genes (named AvCAT1 to AvCAT10; Table 1) were identified in oats.Furthermore, an unrooted phylogenetic tree was constructed using MEGA 11 software with different CAT proteins from Arabidopsis thaliana (3 proteins), T. aestivum (10 proteins), T. turgidum (6 proteins), Nicotiana plumbaginifolia (3 proteins); and Oryza sativa ssp Japonica (4 proteins) (Figure 2).As represented by the phylogenetic tree, thirty-six CAT genes were divided into three different classes (class I, class II, and class III), with an extra group formed exclusively by OsCATD.The dicotyledonous CAT proteins identified in A. thaliana and N. plumbaginifolia were clustered in the same group together with oat CAT belonging to the second group (AvCAT4/5/6 and 7), OsCATB and TaCAT3 (A1/A2/B/U) and no CAT from T. turgidum suggesting a related relationship between those proteins.The other two groups (groups 1 and 3) are formed exclusively by monocotyledonous CATs.Group 1 is composed of Avena CAT group 3 (AvCAT8, 9 and 10), TaCAT2 (genome A/B/D), OsCATA/D, and TdCAT2/3/4 and 6, which suggests that those CAT proteins could share the same functions in plants.The last group is formed by Avena CAT class 1, TdCAT1/5, TaCAT1A/B/D, and OsCATC (Figure 2).A phylogenetic tree was produced using the ten AvCAT proteins identified in the allohexaploid genome to investigate the evolutionary relationship between oat CAT proteins (Figures 1A and 2).The phylogenetic tree showed that AvCAT proteins are subdivided into three clusters using two servers (Figure 1B; Supplemental Figure S1).AvCAT1, AvCAT2, and AvCAT3 proteins are clustered in the same group (group 1).The second group is composed of AvCAT4, AvCAT5, AvCAT6, and AvCAT7 proteins, whereas the third group is formed by AvCAT8, AvCAT9, and AvCAT10 proteins.Furthermore, an unrooted phylogenetic tree was constructed using MEGA 11 software with different CAT proteins from Arabidopsis thaliana (3 proteins), T. aestivum (10 proteins), T. turgidum (6 proteins), Nicotiana plumbaginifolia (3 proteins); and Oryza sativa ssp Japonica (4 proteins) (Figure 2).As represented by the phylogenetic tree, thirty-six CAT genes were divided into three different classes (class I, class II, and class III), with an extra group formed exclusively by OsCATD.The dicotyledonous CAT proteins identified in A. thaliana and N. plumbaginifolia were clustered in the same group together with oat CAT belonging to the second group (AvCAT4/5/6 and 7), OsCATB and TaCAT3 (A1/A2/B/U) and no CAT from T. turgidum suggesting a related relationship between those proteins.The other two groups (groups 1 and 3) are formed exclusively by monocotyledonous CATs.Group 1 is composed of Avena CAT group 3 (AvCAT8, 9 and 10), TaCAT2 (genome A/B/D), OsCATA/D, and TdCAT2/3/4 and 6, which suggests that those CAT proteins could share the same functions in plants.The last group is formed by Avena CAT class 1, TdCAT1/5, TaCAT1A/B/D, and OsCATC (Figure 2).
Subsequently, the conserved domain of candidate AvCAT protein sequences was analyzed (Figure 1B).The multiple alignments performed using the Muscle algorithm showed that AvCAT protein sequences are conserved (Supplemental Figure S2).Based on the domain analysis, all identified AvCAT proteins contained one catalase core domain (PF00199, Catalase) and one catalase immune-responsive domain (PF06628, Catalase-rel), forming the fundamental catalase domains.The catalase-rel domain was an immune-responsive amphipathic octa-peptide that was found in the C-terminal of CATs.In addition, despite their different sizes, all CAT proteins have the same conserved domains, such as pfam00199 and pfam06628 domains.Moreover, as found in typical CAT proteins, all identified AvCAT proteins harbor a conserved catalase activity motif (CAM: FARERIPERVVHARGAS) site, which also presented the conserved Histidine residue at position 65 (Supplemental Figure S2).In addition, a conserved heme-binding site (HBS: RVFAYGDTQ) with a conserved Tyrosine (Y350) is also conserved in all AvCAT proteins (Supplemental Figure S2).Finally, a PTS1-like motif (QKL/I/V) was also mapped in AvCAT1/2/3/5/6/7, whereas AvCAT4 presents the CSS motif and AvCAT8/9/10 presents the MKV motif in their sequences (Supplemental Figure S2).Moreover, the histidine residue is conserved in all identified proteins (Supplemental Figure S2).Subsequently, the conserved domain of candidate AvCAT protein sequences was analyzed (Figure 1B).The multiple alignments performed using the Muscle algorithm showed that AvCAT protein sequences are conserved (Supplemental Figure S2).Based on the domain analysis, all identified AvCAT proteins contained one catalase core domain (PF00199, Catalase) and one catalase immune-responsive domain (PF06628, Catalase-rel), forming the fundamental catalase domains.The catalase-rel domain was an immune-responsive amphipathic octa-peptide that was found in the C-terminal of CATs.In addition, despite their different sizes, all CAT proteins have the same conserved domains, such as pfam00199 and pfam06628 domains.Moreover, as found in typical CAT proteins, all identified AvCAT proteins harbor a conserved catalase activity motif (CAM: FARE-RIPERVVHARGAS) site, which also presented the conserved Histidine residue at position 65 (Supplemental Figure S2).In addition, a conserved heme-binding site (HBS: In the second step, we used the Multiple Em for Motif Elicitation (MEME) database (version 5.5.1) to map the putative conserved motifs in the identified AvCAT proteins.As shown in Figure 1C, thirteen motifs were identified.Interestingly, those motifs are present in all identified proteins except AvCAT9, which lacks motif 9 (presented by dark purple boxes).Moreover, to understand the evolution of AvCAT genes, analyses of the exon-intron organization of AvCAT genes was performed.The result of the AvCAT gene structure showed that the numbers of exons varied between two and nine, with the lowest numbers of exons in AvCAT10 and the highest number in AvCAT6 (Table 1; Figure 1D).AvCAT8 and AvCAT9, which belong to the same cluster, present three exons.AvCAT3 harbors five exons, whereas AvCAT1 and 2, belonging to the same cluster, harbor six exons.The other catalases belonging to the same group harbor seven exons in AvCAT4, eight exons in AvCAT5, and seven and nine exons in AvCAT6.The genomic features of identified AvCAT genes are detailed in Table 1.

Gene Distribution of Catalase Genes in Oat
The distribution of CAT genes in allohexaploid oats was observed on different chromosomes.In fact, two CAT-encoding genes are located in chromosome 1 (genomes A and D); one CAT gene is located in chromosome 2D; two CAT-encoding genes are located in chromosome 4 (genomes A and C); two CATs encoding genes are located in chromosome 6 and three CAT encoding genes are located in chromosome 7 (genome D) (Table 1, Figure 3).
(version 5.5.1) to map the putative conserved motifs in the identified AvCAT proteins.As shown in Figure 1C, thirteen motifs were identified.Interestingly, those motifs are present in all identified proteins except AvCAT9, which lacks motif 9 (presented by dark purple boxes).Moreover, to understand the evolution of AvCAT genes, analyses of the exon-intron organization of AvCAT genes was performed.The result of the AvCAT gene structure showed that the numbers of exons varied between two and nine, with the lowest numbers of exons in AvCAT10 and the highest number in AvCAT6 (Table 1; Figure 1D).AvCAT8 and AvCAT9, which belong to the same cluster, present three exons.AvCAT3 harbors five exons, whereas AvCAT1 and 2, belonging to the same cluster, harbor six exons.The other catalases belonging to the same group harbor seven exons in AvCAT4, eight exons in AvCAT5, and seven and nine exons in AvCAT6.The genomic features of identified AvCAT genes are detailed in Table 1.

Gene Distribution of Catalase Genes in Oat
The distribution of CAT genes in allohexaploid oats was observed on different chromosomes.In fact, two CAT-encoding genes are located in chromosome 1 (genomes A and D); one CAT gene is located in chromosome 2D; two CAT-encoding genes are located in chromosome 4 (genomes A and C); two CATs encoding genes are located in chromosome 6 and three CAT encoding genes are located in chromosome 7 (genome D) (Table 1, Figure 3).

AvCAT Proteins Characteristics
All identified proteins are stable except AvCAT3 and AvCAT5.In addition, the secondary (2D) structure of AvCAT proteins was predicted using the SOPMA program.Interestingly, five proteins (AvCAT1/2/3/6 and 7) present a high aliphatic index (>70), which suggests that those proteins are thermostable over a wide temperature range.Moreover, AvCAT proteins presented small, disordered regions in their structures located at the Cterminal part of the proteins, except for AvCAT4, where the disordered region was located

AvCAT Proteins Characteristics
All identified proteins are stable except AvCAT3 and AvCAT5.In addition, the secondary (2D) structure of AvCAT proteins was predicted using the SOPMA program.Interestingly, five proteins (AvCAT1/2/3/6 and 7) present a high aliphatic index (>70), which suggests that those proteins are thermostable over a wide temperature range.Moreover, AvCAT proteins presented small, disordered regions in their structures located at the Cterminal part of the proteins, except for AvCAT4, where the disordered region was located in the N-terminal part (Table 2).All identified proteins revealed alpha helix, beta turns, extended strand, and random coil.These structures were represented by small lines of different colors (Supplemental Figure S3; Table 2).
Interestingly, the organization of these secondary structures of AvCAT proteins differed.In fact, the 2D structure of AvCAT1, 2, 4, and 8 was very similar despite the fact that they belong to different sub-classes, as revealed by the phylogenetic analysis.AvCAT4 differs slightly from those proteins in the C-terminal part.Despite their similarity in the CDS organization and the motifs identified in their structures, AvCAT8 slightly differs from AvCAT9 and AvCAT10 2D structures, whereas AvCAT9 and AvCAT10 structures were more similar.The same result was obtained for AvCAT5 and AvCAT7.Finally, the AvCAT3 and AvCAT6 structures differ from each other and the other proteins (Supplemental Figure S3).Random coil accounted for a large proportion (47-54%) of all identified CAT proteins.The second was alpha-helix (26-30%), and those components were concentrated in the N-terminal region of the proteins.Beta turns formed the smallest proportion (4-6%) of secondary structures, whereas the extended strands counted between 13 and 16% (Table 2).In addition, the Alphafold server was used to predict the 3D structures of the proteins (Figure 4).The AvCAT tridimensional protein structures have some differences from each other, confirming the results already described by the 2D structure.In fact, AvCAT1, 2, 4, and 8 was very similar.AvCAT9 and AvCAT10 structures were also very similar, as revealed by the 2D structure-the same for AvCAT5 and AvCAT7.Finally, the AvCAT3 and AvCAT6 structures differ from each other and the other proteins (Figure 4).Interestingly, all proteins' GRAVY index (grand average of hydropathy) is negative, suggesting that those proteins are hydrophobic.Finally, all identified proteins have a high percentage of random coils (Table 3).CAT proteins do not have a signal peptide site in their structures, except for AvCAT5 (Table 3), as revealed by the Protter server.Finally, a glycosylation site was identified for all oat CAT proteins except for AvCAT5, which presented two glycosylation sites, and AvCAT6, which does not have a glycosylation site.These sites are located at different parts of the proteins (Table 3).Additionally, using NetPhos-3.1:https://services.healthtech.dtu.dk/services/NetPhos-3.1/, AvCAT proteins were analyzed to count the number of phosphorylated sites in the proteins.As expected, all AvCAT proteins are phosphorylable as the number of identified phosphorylation sites varies from 29 in AvCAT6 to 50 in AvCAT5, which suggests that protein phosphorylation is important for AvCAT protein activities (Table 3).In addition, no transmembrane region was found in all identified AvCAT proteins except for AvCAT5.Interestingly, all proteinsʹ GRAVY index (grand average of hydropathy) is negative, suggesting that those proteins are hydrophobic.Finally, all identified proteins have a high percentage of random coils (Table 3).CAT proteins do not have a signal peptide site in their structures, except for AvCAT5 (Table 3), as revealed by the Protter server.Finally, a glycosylation site was identified for all oat CAT proteins except for AvCAT5, which presented two glycosylation sites, and AvCAT6, which does not have a glycosylation site.These sites are located at different parts of the proteins (Table 3).Additionally, using NetPhos-3.1:https://services.healthtech.dtu.dk/services/NetPhos-3.1/, AvCAT proteins were analyzed to count the number of phosphorylated sites in the proteins.As expected, all AvCAT proteins are phosphorylable as the number of identified phosphorylation sites varies from 29 in AvCAT6 to 50 in AvCAT5, which suggests that protein phosphorylation is important for AvCAT protein activities (Table 3).In addition, no transmembrane region was found in all identified AvCAT proteins except for AvCAT5.

In Silico Analysis of AvCAT Proteins
On the other hand, the subcellular localization of AvCAT proteins was performed using the Wolf PSORT online server.As shown in Figure 5, AvCAT proteins presented different subcellular localizations.In fact, no catalase protein was identified in the extracel-Plants 2023, 12, 3694 lular compartment, endoplasmic reticulum, and plasmatic membrane.AvCAT1, AvCAT2, and AvCAT4 are essentially peroxisomal proteins.AvCAT3 could be located in the mitochondria and chloroplasts.AvCAT5 and AvCAT7 are chloroplastic.AvCAT6 could be found in the peroxisome, mitochondria, and the cytoplasm.AvCAT8 and AvCAT9 are predominantly located in the cytoplasm but could also be mitochondrial and peroxisomal, whereas AvCAT10 is predicted to be a cytoplasmic, peroxisomal, and mitochondrial protein with a small probability of a nucleic localization (Figure 5).

In Silico Analysis of AvCAT Proteins
On the other hand, the subcellular localization of AvCAT proteins was performed using the Wolf PSORT online server.As shown in Figure 5, AvCAT proteins presented different subcellular localizations.In fact, no catalase protein was identified in the extracellular compartment, endoplasmic reticulum, and plasmatic membrane.AvCAT1, AvCAT2, and AvCAT4 are essentially peroxisomal proteins.AvCAT3 could be located in the mitochondria and chloroplasts.AvCAT5 and AvCAT7 are chloroplastic.AvCAT6 could be found in the peroxisome, mitochondria, and the cytoplasm.AvCAT8 and AvCAT9 are predominantly located in the cytoplasm but could also be mitochondrial and peroxisomal, whereas AvCAT10 is predicted to be a cytoplasmic, peroxisomal, and mitochondrial protein with a small probability of a nucleic localization (Figure 5).

Identification of CaM Binding Domains
To search whether identified oat CAT proteins harbor a calmodulin-binding domain, we analyzed the structure of the identified proteins using the calmodulin target database.As revealed in Table 4, all identified AvCAT proteins harbor at least three putative CaMBDs located at different parts of the proteins.All identified CAT proteins harbor an IQ motif (Table 4).The biological significance of such domains remains unclear.

Identification of CaM Binding Domains
To search whether identified oat CAT proteins harbor a calmodulin-binding domain, we analyzed the structure of the identified proteins using the calmodulin target database.As revealed in Table 4, all identified AvCAT proteins harbor at least three putative CaMBDs located at different parts of the proteins.All identified CAT proteins harbor an IQ motif (Table 4).The biological significance of such domains remains unclear.

Gene Ontology (GO) Term Distribution of A. sativa Catalase
To identify the biological process and the molecular functions of the different isolated proteins, the Pannzer2 tool was used.The results, represented in Figure 6, showed that all identified proteins have a catalase activity and present heme/metal binding motifs.Additionally, AvCAT proteins present a protein-binding function.In addition, AvCAT1, 2, and 3 are structural constituents of ribosomes, whereas AvCAT1, 2, 3, and 5 have 5S rRNA binding functions (Figure 6).Interestingly, oat CATs have different functions.In fact, all identified AvCAT control the cellular oxidant detoxification and hydrogen peroxide catabolic process.Moreover, those genes are implicated in response to hormones and different abiotic stresses.AvCAT4, 5, 6, 7, 8, 9, and 10 are involved in cell response to ROS whereas, AvCAT1, 2, and 3 are involved in response to oxygen-containing substances, oxidative stress, intracellular nitric oxide homeostasis, Hydrogen peroxide biosynthesis processes and implicated in protein nitrosylation.In addition, AvCAT8, 9, and 10 control plant response to inorganic substances and salt stress.AvCAT4/5/6 and 7 control plant response to heat and, together with AvCAT4, 5, and 6, modulate plant response to cadmium.AvCAT4, 5, 6, 7, 8, 9, and 10 control the circadian rhythm of the plants and their response to alcohol stress.Finally, AvCAT4, 5, 6, 7, 9, and 10 ensure plant response to acid chemicals (Figure 6).

In Silico Analysis of Cis-Elements
To further investigate the cis-elements of different CAT genes, the 2 kb 5′ upstream region of the 10 AvCAT genes was studied using the PlantCARE database.Our results showed the presence of some basic core components as well as the different cis-acting elements that could be divided into three categories: environmental stress-related elements (like drought inducibility, light-responsive and low temperature-responsive), an-

In Silico Analysis of Cis-Elements
To further investigate the cis-elements of different CAT genes, the 2 kb 5 upstream region of the 10 AvCAT genes was studied using the PlantCARE database.Our results showed the presence of some basic core components as well as the different cis-acting elements that could be divided into three categories: environmental stress-related elements (like drought inducibility, light-responsive and low temperature-responsive), anoxia or anaerobic induction elements, and hormone-responsive elements (such as Me-Jasmonic acid (MeJA), salicylic acid (SA), auxin, Gibberelline, and abscisic acid-responsive (ABRE)) and development-related elements (such as cell cycle regulation and meristem expression) (Figure 7).Different identified elements, such as ABRE and MeJA-response elements, are crucial for abiotic stress response.G-box and ABRE elements are common for all identified AvCAT genes (Figure 7).Moreover, all AvCAT genes are implicated in plant response to light, but only AvCAT4 and AvCAT7 are implicated in plant response to low temperatures, whereas AvCAT3 is the only CAT gene that presents responsive elements to SA in its promoter.Thus, CAT genes from oats belonging to the same sub-group could have various modes of action, and genes of different classes may work together.

Expression Analysis of AvCATs in Different Tissues/Organs and under Different Stress Conditions
To investigate the biological functions of the CAT gene family in the allohexaploid oat plant further, we investigated the transcriptome data of different tissues/organs.In fact, in our analysis, we examined the expression pattern of AvCAT genes in three organs of oats (stems, roots, and shoots) at 10 days-old stage at normal conditions.As seen in Figure 8, all

Expression Analysis of AvCATs in Different Tissues/Organs and under Different Stress Conditions
To investigate the biological functions of the CAT gene family in the allohexaploid oat plant further, we investigated the transcriptome data of different tissues/organs.In fact, in our analysis, we examined the expression pattern of AvCAT genes in three organs of oats (stems, roots, and shoots) at 10 days-old stage at normal conditions.As seen in Figure 8, all AvCATs have a constitutive expression in all organs.Remarkably, the AvCAT3 gene, which belongs to group I, showed a lower expression than other genes of group I, especially in roots and leaves, whereas AvCAT1 presented a higher expression level (Figure 8).In the second step, we chose 3 AvCAT genes representing three different AvCAT gene classes, AvCAT2, AvCAT4, and AvCAT8, to study their expression under different stress conditions.Under heat stress conditions (37 °C), AvCAT2 had no variation in gene expression, and the transcription level remained unchanged (Figure 9A).Interestingly, AvCAT4 and AvCAT8 gene expression started to increase after 1 h of stress application to reach their maximum after 12 h before declining after 24 h of stress (Figure 9B,C).
Under cold stress conditions, AvCAT2 and AvCAT8 did not present any modification of gene expression levels in contrast to AvCAT4, which showed an increase in its transcript level (Figure 9D-F).Such results showed the importance of AvCAT4 in plant response to heat and cold stress and the role of AvCAT8 in heat stress.Moreover, AvCAT2, a group I catalase, is not implicated in plant response to extreme temperature stress.
Our results suggest that AvCAT2 is not involved in plant defense against heat and cold stresses, whereas AvCAT4 is crucial in plant response to both stresses.Moreover, AvCAT8 is involved in heat stress response (Figure 9).

Relative expression level
. RT-qPCR expression analysis of AvCAT genes under normal conditions.AvCAT gene expression of groups 1 (A), 2 (B), and 3 (C) was analyzed under normal conditions using tissues from roots, leaves, and stems.
In the second step, we chose 3 AvCAT genes representing three different AvCAT gene classes, AvCAT2, AvCAT4, and AvCAT8, to study their expression under different stress conditions.Under heat stress conditions (37 • C), AvCAT2 had no variation in gene expression, and the transcription level remained unchanged (Figure 9A).Interestingly, AvCAT4 and AvCAT8 gene expression started to increase after 1 h of stress application to reach their maximum after 12 h before declining after 24 h of stress (Figure 9B,C).
Under cold stress conditions, AvCAT2 and AvCAT8 did not present any modification of gene expression levels in contrast to AvCAT4, which showed an increase in its transcript level (Figure 9D-F).Such results showed the importance of AvCAT4 in plant response to heat and cold stress and the role of AvCAT8 in heat stress.Moreover, AvCAT2, a group I catalase, is not implicated in plant response to extreme temperature stress.Under ABA stress conditions, AvCAT2 was rapidly activated in all investigated tissues with a maximum induction in leaves.Moreover, the AvCAT2 expression level remains elevated after 24 h of stress application (Figure 10).The same effect was observed with AvCAT4.In contrast, AvCAT8 was down-regulated in the presence of ABA, suggesting that AvCAT8 could be a negative regulator of plant response to ABA.Our results suggest that AvCAT2 is not involved in plant defense against heat and cold stresses, whereas AvCAT4 is crucial in plant response to both stresses.Moreover, AvCAT8 is involved in heat stress response (Figure 9).
Under ABA stress conditions, AvCAT2 was rapidly activated in all investigated tissues with a maximum induction in leaves.Moreover, the AvCAT2 expression level remains elevated after 24 h of stress application (Figure 10).The same effect was observed with AvCAT4.In contrast, AvCAT8 was down-regulated in the presence of ABA, suggesting that AvCAT8 could be a negative regulator of plant response to ABA.

Discussion
In addition to model plants like A. thaliana [45], rice [11], and N. tabacum [20], genomewide identification of the CAT gene family has also been reported in several other crops, including durum wheat [16], G. hirsutum, G. max, and G. barbadense [17], H. vulgare [46], Z. mays [47], pumpkin [48], bread wheat ( [12], C. sativus [19], Rapeseed [49] and so on.Recently, sixteen CAT genes have been discovered and cloned from Saccharum spontaneum [18], five from sugarcane hybrids [28], and two CAT genes in E. arundinaceus [50].In the present work, a total of 10 CATs encoding genes were identified by bioinformatics analysis (Table 1).According to their structure/functions, it has been shown that plant CAT genes are generally divided into three different groups related to photosynthetic, vascular, and reproductive functions [19,48].The same observation was also described here; oat CAT proteins were subdevised into three groups (Figure 1A).The same result was also described in cucumber [19]; T. durum [16]; and T. aestivum [51].Generally, prokaryotic and eukaryotic catalases were also classified into three classes: A subgroup of bacteria and tiny plant catalase subunits were found in Clade 1.The third clade includes small subunit catalases from bacteria, fungi, protists, animals, and plants, while the second clade includes a subset of bacteria and large subunit catalases from fungi [52].Thus, such findings strongly support the reliability of the group classifications in our study.
Catalase sequence alignment suggests a high similarity percentage (>95%) (Supplemental Figure S3) in each class.The similarity of two protein sequences in each class is very high, as previously shown for CAT identified from T. durum [16] and N. plumbaginifolia [53].Moreover, the chromosomal localization of AvCAT genes was also investigated.The 10 genes were located in 7 different chromosomes.In fact, AvCAT7 and AvCAT10 genes were mapped in Ch6C, AvCAT4; AvCAT5 and AvCAT6 were located in Ch7D, whereas the other genes were located in the other chromosomes (Ch1D, Ch4A, Ch4C, and Chr2D) (Table 1, Figure 3).In T. durum, six genes were located on three different chromosomes: chromosome 6B (TdCAT3, TdCAT4, and TdCAT6); chromosome 4B (TdCAT1 and TdCAT5) and on chromosome 6A (TdCAT2B) [16].In bread wheat, 9 out of the 10 TaCAT genes were mapped onto the distal regions of the arms of eight different wheat chromosomes [12].
Plant CATs have two well-conserved domains.A typical plant CAT enzyme is tetrameric, and each subunit contains different conserved sequences at the catalytic site.Moreover, those proteins present a catalase activity motif (CAM) with a conserved histidine at position 65 (FARERIPERVVHARGAS), a conserved peptide sequence (PTS1) (S/E/C-K/R/H-L) at the carboxyl terminus as well as heme binding sites (HBS) containing a conserved tyrosine at position 350 (RVFAYGDTQ).The PTS1 is nine amino acids in length from the carboxy terminus and may be able to recognize the peroxisome [54].In addition, Kamigaki et al. [55] reported the discovery of another conserved PTS1-like motif (QKL/I/V).Cucumber has four CsCAT proteins, which also present three conserved amino acids-His, Asn, and Tyr-a catalytic site-FDRERIPERVVHAKGAGA-and a conserved heme-ligand signature sequence-RLFSYNDTH.The same characteristics were also found in CAT1 isolated from durum wheat [56].
Protein Subcellular location is a crucial biological characteristic of proteins [57], which allows scientists to understand the mechanisms controlling protein activities.Subcellular localization of different catalase proteins was investigated in different species such as in Arabidopsis (the peroxisomes), in rice (peroxisomes and cytoplasm) [11], in T. turgidum and T. monococcum (TdCAT1 and TmCAT1, respectively were located in the peroxisome) [56], in bread wheat TaCAT2A/B was localized in the cytoplasm and the nucleus [12].In this study, a PTS1-like motif (QKL/I/V) was mapped in 6 proteins (AvCAT1/2/3/5/6/7) but absent in the other proteins (Supplemental Figure S2).Interestingly, in silico analyses of those proteins, it was revealed that AvCAT1 and AvCAT2 are peroxisomal proteins.Despite the absence of a PTS1-like motif in its structure, AvCAT4 (CSS motif instead of QKL/I/V motif) is predicted to be also peroxisomal.The mechanism of peroxisomal localization of AvCAT4 remains unclear.AvCAT8 and AvCAT9 could be located in the cytoplasm, mitochondria, and peroxisome, whereas AvCAT10 is predicted to be a cytoplasmic, peroxisomal, and mitochondrial protein with a small probability of a nucleic localization.Those proteins have an MKV motif, not a PTS1-like motif (Supplemental Figure S2).Despite the presence of the PTS1-like motif, AvCAT3 is located in the nucleus, mitochondria, and cytoplasm but not in the peroxisome (Figure 5).The same finding was for AvCAT6, which has a small probability of being located in the peroxisome (Figure 5).Such findings suggest the importance of different localizations of CAT proteins in plants to eliminate the toxic H 2 O 2 compounds.
Moreover, the result of the AvCAT gene structure showed that the numbers of exons varied between two and 9, with the lowest number of exons in AvCAT10 and the highest number in AvCAT10.In T. durum, the number of exons varies from 3 to 7 [16], whereas in G. hirsutum, the structure of the seven identified genes varies between seven and nine.Identified AvCAT proteins are hydrophobic (negative GRAVY index), and five proteins (AvCAT1/2/3/6/7) are thermostable (aliphatic index > 70%) (Table 2).Interestingly, the majority of identified CAT proteins in all investigated species have a negative GRAVY index, suggesting that those proteins are thermostable.Furthermore, AvCAT proteins share small, disordered regions in their sequences, and no signal peptide was mapped except for AvCAT5 (Table 2).The 2D and 3D protein structures were also studied using SOPMA and Alphafold servers, respectively.Interestingly, the structures of identified AvCAT proteins were predominantly formed by random coils (approximately half of the protein structures).Such results were also shown for durum wheat [16] and tobacco catalase proteins [20].The AvCAT 3D models presented a variation in their structural conformation.The binding pockets play a crucial role in the protein interaction and binding sites.According to the CASTp 3.0 analysis, molecular pockets were identified on all candidates.The top three predicted pockets with the largest volume are indicated as pink, purple, and green, respectively (Figure 4).These giant pockets exhibited in the AvCAT protein structure may be related to the highest number of candidates who could bind to their atoms.Therefore, different molecular functions may be associated with these catalases in A. sativa.
For the diversity of protein functions in plant cell signaling, posttranslational modifications (PTMs) are significant regulators.Protein phosphorylation is a significant and well-studied PTM that affects the functionality of numerous receptors and essential elements in cellular signaling.Protein kinases and protein phosphatases, respectively, catalyze the dynamic and reversible protein phosphorylation that mostly takes place on serine (Ser) and threonine (Thr) residues in plants.In fact, many physiological processes are controlled by protein phosphorylation, such as iron uptake as Fe homeostasis is controlled by protein phosphorylation [58], nitrogen, phosphorus, and potassium uptake in plants [59], plant immunity [60], anthocyanin accumulation in apple fruits [61] and so on.As shown in Table 3, the number of phosphorylated sites in AvCAT proteins varies from 29 in AvCAT6 to 50 in AvCAT5, suggesting the importance of phosphorylation in AvCAT protein activities.Recently, it has been shown that TdCAT proteins from durum wheat presented different phosphorylation sites [16].Moreover, TdCAT1 activity depends on the phosphorylation status of the protein.In fact, treatment of TdCAT1 by phosphatase inhibited the catalytic activity of the protein [14].Interestingly, it has been recently shown that TdCAT1 proteins could be phosphorylated in vitro in the presence of wheat Mitogen-Activated Protein Kinase protein (TMPK3) and that the presence of TMPK3 enhanced the catalytic activity of the catalase [62].In the current work, in silico analyses showed that AvCAT proteins could be phosphorylated by MAPKs.The importance of such results should be investigated in vivo to study the role of each phosphorylation residue in plant growth/development as well as plant response to different stresses.
One of eukaryotic proteins' most prevalent posttranslational modifications is glycosylation [63].All identified CAT proteins harbor putative N-glycosylation site in their structures except for AvCAT6, which do not contain any putative glycosylation site, and AvCAT5, which harbors two putative glycosylation sites as revealed by the Protter database (Table 3).In eukaryotes, this modification regulates different signaling pathways implicated in the modulation of plant response to different stresses.In plants, the procedure of Nglycosylation keeps the chloroplast-located protein known as CAH1 stable, which plays an important function in controlling photosynthetic efficiency.The folding and transportation of proteins both benefit from N-glycosylation.Glycosylation is also crucial for stomata development.In fact, a mutation in the STT gene enhances transpiration in plants, leading to an important water loss in plants and an abnormal stomatal distribution.Thus, plants are more sensitive to drought stress [64].Moreover, it has been shown that mutations in the N-glycosylation pathway genes alg3-3 and cgl1-1 in Arabidopsis result in a clear reduction in photosynthesis [65].In addition, stt3a mutant plants are characterized by cell death inhibition in the presence of bir1 and bak1 serk4 mutations [66].Finally, under-glycosylation of a β-glucosidase protein (AtBG1) inhibits ABA and Auxin biosynthesis.In fact, AtBG1 controls the transformation of conjugated IAA/ABA to active hormones, suggesting that N-glycosylation is important for stomatal development and controlling the endogen level of active hormones in response to abiotic stresses [66].
Calmodulins (CaMs), the most relevant calcium sensors, are small acidic proteins highly conserved by eukaryotes [67].Those sensors perceive small changes in intracellular Ca 2+ levels [68] to ensure plants' response to different plant growth cascades and response to biotic and abiotic stresses [69,70] by fixing a large number of ligands such as transcription actors [70], MAP Kinase Phosphatase [71], pathogen-related protein (PR-1) [72].It has been shown that durum wheat harbors 6 CATs encoding genes.All identified durum wheat CAT proteins harbor at least three conserved CaMBDs located at different portions of the proteins [16].Moreover, we have recently characterized a conserved CaM binding domain (CaMBD) located at the C-terminal portion of the TdCAT1 protein [17] and in many other proteins such as Arabidopsis [73], potato [74], and sweet potato [75] TdCAT1/CaM interaction (in presence of Ca 2+ ions) enhances the catalytic activity of the protein in Calcium dependent manner.In the present work, all CAT proteins harbor at least three calmodulin-binding domains.Those domains are located at different portions of the proteins, as previously shown for durum wheat [17] (Table 4).The results suggest that the AvCAT gene family has conserved catalase structural domains, presents catalytic functions, and can share the same functions as other identified plant CATs.
Gene ontology analysis was also performed.The results confirmed that all identified proteins presented catalase activity and heme/metal binding motifs, suggesting the importance of Heme and other cations in protein catalase activity.In fact, it has been recently demonstrated that durum wheat catalase activity (TdCAT1) was stimulated in the presence of Fe 2+ and other cations (Mn 2+ ; Mg 2+ ; Ca 2+ ; Zn 2+ ; Cu 2+ ).Interestingly, the catalase activity of TdCAT1 increased gradually with increasing concentrations of cations in the medium, with the most important effect being enregistered in the presence of Fe 2+ and Mn 2+ [13].In addition, those proteins can interact with other proteins, as previously described, such as the CaM/Ca 2+ complex [13] and protein kinases [62].In addition, AvCAT1/2 and 3 are structural constituents of ribosomes, whereas AvCAT1/2 /3 and 5 have 5S rRNA binding functions (Figure 6).
Interestingly, oat CATs have different functions.In fact, all identified AvCAT control the cellular oxidant detoxification and hydrogen peroxide catabolic process.The same result was observed in durum wheat [16].Moreover, those genes are implicated in response to hormones and different abiotic stresses.AvCAT4/5/6/7/8/9 and 10 are involved in cell response to ROSs, whereas AvCAT1/2 and three are involved in response to oxygencontaining substances, oxidative stress, intracellular nitric oxide homeostasis, Hydrogen peroxide biosynthesis processes and implicated in protein nitrosylation.In addition, Av-CAT8/9 and 10 control plant response to inorganic substances and salt stress.AvCAT4/5/6 and 7 control plant response to heat, and together with AvCAT4/5 and 6, they modulate plant response to cadmium-avCAT4/5/6/7/8/9 and 10 control the circadian rhythm of the plants and their response to alcohol stress.Finally, AvCAT4/5/6/7/9 and 10 ensure plant response to acid chemicals (Figure 6).
Depending on recent bibliography research, no research has been carried out to investigate the cis-acting elements of AvCAT gene promoters.To further understand ciselements of AvCAT genes, the 2 kb 5 upstream region of the 10 AvCAT genes was analyzed using the PlantCARE database.Our results showed that all AvCAT genes have some basic core components.Moreover, two elements (G-box (Sp1) and ABA-response element (ABRE), which are crucial for plant response to abiotic stress, were common to all identified AvCAT genes (Figure 6).Such results were also observed in bread and durum wheat [12,16].AvCAT genes are responsive to different abiotic stress and developmental events.For example, all AvCAT genes are implicated in plant response to light, whereas AvCAT3 contains ciselements associated with SA responses.The promoters of AvCAT1/2/4/8 and 9 specifically contained an element related to meristem expression, suggesting that these genes may be related to meristem development.AvCAT1/3/5/6 and 10 are responsive to Gibberellic acid, whereas AvCAT4/5/6/7/8/9 and 10 are responsive to auxin.Moreover, AvCAT4 and AvCAT7 are implicated in plant response to low temperatures, and AvCAT4/5/6/7/8/9 and 10 are responsive to drought stress.Moreover, an MYB-binding site (MBS) was found in the promoter region of oat CAT genes, which belong to classes II and III, suggesting that those AvCATs could be regulated by the MYB transcription factor.All those findings suggest that AvCAT genes can be involved in plant maturation/growth and cell differentiation by acting as ROS regulators.Moreover, our findings suggest that AvCAT genes of the same class may have different modes of action and that genes of different classes may work together, as previously shown for TaCAT and TdCAT genes [12,16].
Previous research demonstrated that the CAT gene expression patterns differed in many tissues and during various growth and development phases.In banana peel, MaCAT2 was down-regulated during fruit maturation [29].Moreover, most of the 14 BnCATs were strongly expressed in the leaf, stem, and silique of B. napus.While BnCAT1, BnCAT3, and BnCAT9 did not express or were expressed at low levels in the majority of tissues, BnCAT4 and BnCAT10 were expressed at a high level [21].Su et al. [28] and Sun et al. [76] observed that in sugarcane, ScCAT1 and ScCAT2 expressed constitutively in leaf, stem epidermal, root, stem pith, and bud, with the highest levels of ScCAT1 and ScCAT2 expression in stem epidermal and bud, respectively.CAT is crucial for growth and development, oxidative senescence, and a protective response to environmental stress in plants.Light, temperature, salt, drought, heavy metals, plant hormones, and pathogens all have an impact on CAT activity [77].Alam and Ghosh [11] demonstrated that in A. thaliana, AtCAT1 expression increased in response to oxidative, drought, cold, and heat stresses, AtCAT2 expression increased in response to osmotic, drought, genotoxic, oxidative, and UV-B stresses, and AtCAT3 expression decreased in response to all stresses except cold, osmotic, and UV-B.In bananas, a strong induction of the MaCat2 gene was detected in leaves after plant exposure to low temperatures (10 • C).This induction was low in roots [29].A lower signal was detected in leaves when fruits were treated at 25 • C.However, in the banana fruit pulp, the MaCat2 transcript accumulation was drastically lower at 25 • C and almost undetectable at 10 • C [29].We observed that the MaCat2 transcript increased in response to mechanical damage but not too high-temperature exposure (45 • C) or during fruit maturation [29].In Zea mays, ZaCat2 presented a unique function in eliminating the toxic H 2 O 2 during senescence and regreening [78].
In this study, we investigated plant responses to different abiotic stresses.Our analyses were performed in the presence of two internal controls: ADPR and GAPDH.The ADPR gene has recently been demonstrated to be the best suited for all test-evaluated stresses (cold, drought, salt stress, heat stress), although the tissue determines how it expresses.This gene provided the proper regulation in roots under salt stress and in leaves under drought stress.Additionally, ADPR served as the best internal control in samples subjected to cold and heat shocks.Interestingly, authors advised caution while using actins due to their general instability [79].Our results showed that 3 AvCATs were expressed constitutively in the Saudi variety's roots, stems, and leaves.All AvCAT genes presented a constitutive expression under normal development conditions, with a higher expression level detected for AvCAT1 in all tissues.AvCAT3 gene, a member of group I, displayed a lower level of expression than other group I genes, particularly in roots and leaves, but AvCAT1 displayed a higher level of expression (Figure 8).Moreover, when the AvCAT gene expression patterns were analyzed, it became clear that the AvCAT genes belonging to the same subgroups were similarly expressed as previously expressed in many other species, such as durum wheat [16] and cotton [17].
Plants 2023, 12, 3694 20 of 26 Furthermore, we studied the transcript levels of the 3 AvCATs (AvCAT2, AvCAT4, and AvCAT8) genes under extreme temperatures (heat and cold) and ABA treatments and in three different tissues (Roots, Stems, and Leaves) (Figures 9 and 10).Interestingly, the AvCAT2 gene was upregulated in response to ABA but not to heat and cold stress, whereas the AvCAT4 gene was upregulated under heat, cold, and ABA treatments, and AvCAT8 was upregulated under heat stress conditions but downregulated in response to ABA.In bread wheat, it has been shown that TaCAT3-A1/B/U genes were down-regulated under cold and PEG treatments and induced under heat stress, whereas the TaCAT2 gene was induced under heat treatment.In addition, TaCAT3-A1/B/U was suppressed under cold and Mannitol treatments.In durum wheat, it has been recently demonstrated that TdCAT2 and TdCAT3 were constrictively expressed in all tissues (Roots, Stems, and Leaves) at 10 days old stage, suggesting that these genes could play important roles in controlling durum wheat growth processes [16].The same result was also observed in bread wheat [12].Actually, TaCAT2B and TaCAT3-A1/B/U genes displayed constitutive patterns of expression since they were expressed in a variety of tissues at all developmental stages.Furthermore, in response to heat stress, the latter genes were activated [12].The CaCat2 gene was expressed throughout all of the tissues in hot peppers, whereas the CaCat1 gene was significantly expressed in vascular tissues, and the CaCat3 gene was constitutively expressed in young seedlings and vegetative organs although at a low level [80].
Our findings have opened up new avenues for further research and shed light on the CAT family genes in oats.More research is needed to fully understand the functions of the oat AvCATs genes.

Plant Material
A. sativa L. seeds from the Saudi Arabian variety (cv.AlShinen) were gathered from private fields in Al-Shinen, which is east of Hai'l.Before incubation, 30 mL of a 0.5% sodium hypochlorite solution was applied to nearly 60 seeds and left on for 15 to 20 min.After that, 50 mL of sterile water was used to wash the seeds five times to eliminate the remaining sodium hypochlorite.Under 280 mol m −2 s −1 of photosynthetically active radiation and 16/8 h of light/dark conditions, incubation was carried out at 25 • C. Petri dishes (11 cm wide by 11 cm long by 2.5 cm high) with a sheet of Whatman filter paper and a piece of sponge (to conserve moisture) were used to germinate seeds.Seeds were then placed in a greenhouse.
Ten days after incubation, several stress treatments were applied to the seedlings.The stress treatments employed in this study included distilled water as a control, heat stresses (37 • C), cold stress (4 • C), and ABA treatment (5 mM).Each treatment was carried out three times.Instantly after being harvested, the roots, stems, and leaves were frozen in liquid nitrogen and preserved at −80 • C.

Identification of AvCAT Gene Family
The specific conserved domains of catalase PF00199 and pfam06628 were used as query to run Blast in the genome of A. sativa L in Ensembl Plants release 56 [83]; and HMMER (https://www.ebi.ac.uk/Tools/hmmer/; accessed on 12 May 2023) [84].Thus, ten members were identified in the Avena CAT proteins family.

Promoter Cis-Regulatory Element Analysis of the AvCAT Gene Family
A 2 kb sequence upstream of the translation start site of AvCAT genes was retrieved from the A. sativa L. genome as the promoter sequence obtained from the Ensembl Plants database (https://plants.ensembl.org/info/index.html/;accessed on 11 May 2023) [81] and its cis-regulatory elements were predicted using PlantCARE (http://bioinformatics. psb.ugent.be/webtools/plantcare/html/;accessed on 24 May 2023) [97].

Subcellular and GO Ontology of AvCAT Proteins
Subcellular localization of AvCAT proteins was realized using Wolf PSORTserver (https: //wolfpsort.hgc.jp/;accessed on 24 May 2023) [98] and visualization via Tbtools software v1.123 [81].By PANNZER2 webtool (http://ekhidna2.biocenter.helsinki.fi/sanspanz/;accessed on 26 May 2023), Go ontology (GO) of AvCAT proteins was predicted [99].The RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) was used to independently extract total RNA from the roots, stems, and leaves of A. sativa L. plants (0.5 g of each tissue).After being extracted, RNA was purified from genomic DNA using an RNase-free DNase set from QIAGEN, validated on an agarose gel, quantified, and utilized for firststrand cDNA synthesis using an oligo-dT primer from the GoScript Reverse Transcription System from Promega (Madison, WI, USA).The following ingredients were used in the PCR reactions performed in 10 µL of final volume: 2 µL of cDNA (obtained from 40 ng of RNA that had undergone DNase treatment), 0.5 µL of each primer for the AvCAT genes at a concentration of 10 µM, 5 µL of 2 × SYBR Green I master mix, and 1 µL of RNase-free water.The reactions were constituted of a denaturation step at 95 At the conclusion of cycling, melting curve analysis was utilized to confirm whether there had been a single amplification.The triplicate PCRs' threshold cycle (CT) values were averaged at the experiment's conclusion and utilized for transcript quantification.Here, two internal genes were used as internal expression standards: GAPDH, and ADP-ribosyl cyclase (ADPR), the relative expression ratio of the AvCAT genes was computed [100] in the presence of the following primers: qADPR-F: 5 -CTCATGGTTGGTCTCGATGC-3 and qADPR-R: 5 -ACATCCCAAACAGTGAAGCT-3 for ADPR gene, and qGAPDH-F: 5 -GTTTGGCATCGTTGAGGGTT-3 and qGAPDH-R: 5 -TGCTGCTGGGAATGATGTTG-3 for GAPDH gene.Based on triplicate data, the relative expression level was determined using the 2 −∆∆CT formula, where ∆∆CT = (CT, Target gene CT, Actin) stressed (CT, Target gene CT, Actin).Three separate experiments (three biological replicates) with varying relative expression ratios are given.

Statistical Analysis
Data are reported as mean ± S.E.The results were compared statistically using the Student's t-test, and differences were considered significant at p < 0.05.

Conclusions
In order to prevent cell death, CAT proteins act as crucial barriers by converting the harmful H 2 O 2 into harmless components.The identification and functional characterization of the oat AvCAT genes is yet unknown despite the fact that CAT genes are essential for plant defense against various abiotic stress environments.Here, various in silico analysis approaches were looked at to improve our understanding of the CAT family in A. sativa plants as a whole.Based on the genome of A. sativa L., the ten genes that make up the AvCAT gene family, which has three subfamilies, were discovered in the current study.These genes were spread over seven distinct chromosomes.Other bioinformatic studies demonstrated that AvCAT proteins include highly conserved structural elements like hemebinding domains, peroxisomal targeting signal 1 (PTS1-like domains), Catalase Activity Motifs, Calmodulin binding domains, as well as Catalase-like and Catalase-related motifs.Other bioinformatic analyses demonstrated that the architectures of AvCAT proteins are substantially conserved.Additionally, examination of the AvCAT gene promoters revealed several cis-elements in the region upstream of the AvCAT genes.These components were discovered in durum wheat and may influence how the genes for growth/development, hormones, and stress responses are expressed.

1 .
Bioinformatic Analysis of Catalase Genes in A. sativa L.

Plants 2023 , 27 Figure 1 .
Figure 1.Bioinformatic analysis of AvCAT genes/proteins visualized by Tbtools.(A) A phylogenetic tree produced by MEGA 11 shows the phylogenetic relationship between the identified genes.(B) Identification of conserved catalase domains (catalase-like superfamily and catalase-related superfamily) present in AvCAT proteins as revealed by CDD online tool.(C) Representation of conserved motifs of AvCAT proteins as revealed by MEME server.(D) AvCAT gene's structure.The abscissa in B, C, and D represent the length of the different proteins or genes.The blue rectangle in D represents the CDS of the genes, and the green boxes represent the UTR regions.

Figure 1 .
Figure 1.Bioinformatic analysis of AvCAT genes/proteins visualized by Tbtools.(A) A phylogenetic tree produced by MEGA 11 shows the phylogenetic relationship between the identified genes.(B) Identification of conserved catalase domains (catalase-like superfamily and catalase-related superfamily) present in AvCAT proteins as revealed by CDD online tool.(C) Representation of conserved motifs of AvCAT proteins as revealed by MEME server.(D) AvCAT gene's structure.The abscissa in B, C, and D represent the length of the different proteins or genes.The blue rectangle in D represents the CDS of the genes, and the green boxes represent the UTR regions.

Figure 3 .
Figure 3. Chromosome localization of AvCAT genes.Prediction of AvCAT genes chromosomal localization in A. sativa genome using MG2C v2.0 online tool.The classification was based on their groups I, II, and III.Gene IDs are colored in blue, pink, and green, respectively.

Figure 3 .
Figure 3. Chromosome localization of AvCAT genes.Prediction of AvCAT genes chromosomal localization in A. sativa genome using MG2C v2.0 online tool.The classification was based on their groups I, II, and III.Gene IDs are colored in blue, pink, and green, respectively.

Figure 4 .
Figure 4.The predicted 3D structures of AvCAT were built using the SWISS-MODEL web server, and prediction of the binding pocket of catalase protein in Avena sativa was generated by the CASTp 3.0 online tool.Pockets were visualized from the largest to the smallest pocket with pink, purple, and green colors, respectively.

Figure 4 .
Figure 4.The predicted 3D structures of AvCAT were built using the SWISS-MODEL web server, and prediction of the binding pocket of catalase protein in Avena sativa was generated by the CASTp 3.0 online tool.Pockets were visualized from the largest to the smallest pocket with pink, purple, and green colors, respectively.

Figure 5 .
Figure 5. Prediction of subcellular localization of AvCAT proteins using Wolf PSORT online server and visualization via Tbtools software v1.123.

Figure 5 .
Figure 5. Prediction of subcellular localization of AvCAT proteins using Wolf PSORT online server and visualization via Tbtools software v1.123.

Plants 2023 , 27 Figure 7 .
Figure 7. Frequency of the cis-elements in AvCAT promoters as revealed by Plantcare.

Figure 7 .
Figure 7. Frequency of the cis-elements in AvCAT promoters as revealed by Plantcare.

Plants 2023 , 27 Figure 8 .
Figure 8. RT-qPCR expression analysis of AvCAT genes under normal conditions.AvCAT gene expression of groups 1 (A), 2 (B), and 3 (C) was analyzed under normal conditions using tissues from roots, leaves, and stems.

Figure 9 .
Figure 9. RT-qPCR expression analysis of AvCAT2, AvCAT4, and AvCAT8 genes under heat (A-C) and cold (D-F) stress conditions and using tissues from roots, leaves, and stems.** Indicates values significantly different from the control.Statistical significance was assessed by applying the Student's t-test at p < 0.05.

Figure 9 .
Figure 9. RT-qPCR expression analysis of AvCAT2, AvCAT4, and AvCAT8 genes under heat (A-C) and cold (D-F) stress conditions and using tissues from roots, leaves, and stems.** Indicates values significantly different from the control.Statistical significance was assessed by applying the Student's t-test at p < 0.05.

2023, 12 , 27 Figure 10 .
Figure 10.RT-qPCR expression analysis of AvCAT2 (A), AvCAT4 (B), and AvCAT8 (C) genes under ABA stress treatment in different tissues isolated from roots, leaves, and stems.** Indicates values significantly different from the control.Statistical significance was assessed by applying the Student's t-test at p < 0.05.

Figure 10 .
Figure 10.RT-qPCR expression analysis of AvCAT2 (A), AvCAT4 (B), and AvCAT8 (C) genes under ABA stress treatment in different tissues isolated from roots, leaves, and stems.** Indicates values significantly different from the control.Statistical significance was assessed by applying the Student's t-test at p < 0.05.

4. 9 .
RNA Extraction and Quantitative Real-Time Reverse Transcription PCR (QRT-PCR) • C for 5 min, 40 cycles of 10 s at 95 • C, 20 s at 60 • C, and 30 s at 72 • C, and a melting curve composed of 5 s at 95 • C, 1 min at 65 • C, and 5 min with an increase in temperature from 65 • C to 97 • C. Each stress condition had three biological repetitions, and each sample underwent three technological repetitions.

Table 1 .
Comparison features of CAT genes identified in A. sativa L.

Table 4 .
Number of identified CaMBDs in AvCAT proteins.

Table 4 .
Number of identified CaMBDs in AvCAT proteins.
Figure 6.Go ontology prediction of catalase proteins of A. sativa plant realized by PANNZER2 webtool.